Influenza viral infection continues to cause disproportionate mortality in the elderly (those 65 years of age or older) despite a high level of annual immunization of this population with inactivated trivalent influenza vaccine (TIV). The efficacy and immunogenicity of TIV in the elderly overall is substantially lower than in younger adults, although there is substantial individual variation for reasons that are not well understood. TIV, which contains the hemagglutinin (HA) and neuraminidase (NA) major viral surface proteins and is unadjuvanted, provides protection mainly by inducing neutralizing antibodies to HA. Influenza-specific CD4 T-cell responses induced by TIV may contribute to the antibody response and, in conjunction with CD8 T-cell responses, may enhance viral clearance in the event that infection occurs despite vaccination. It is unclear to what extent the inclusion of potent adjuvants for adaptive immunity, such as cationic lipid/DNA complexes (CLDC) could boost immunogenicity in the elderly to TIV and other vaccines. A phase II human study sponsored by Juvaris Biotherapeutics, Inc., is evaluating the safety, efficacy and dose requirement of a single intramuscular dose of TIV/CLDC for optimal influenza-specific humoral and T-cell responses in 472 elderly men and women. As part of this study, GLP-certified assays of influenza-specific antibody and CD4 and CD8 T-cell responses at baseline and at multiple time points post-vaccination are in progress. The availability of these immunogenicity results, along with multiple aliquots of cryopreserved PBMCs, provides a unique opportunity to determine predictors of vaccine immunogenicity in the elderly in this project. The central hypothesis is that impaired humoral and T-cell immunogenicity to TIV in some of the elderly reflects intrinsic limitations in the signaling of antigen-presenting cells (APCs), B cells, and T cells, and that these limitations are, in turn, the result of alterations of mRNA expression by microRNAs (miRNAs). This hypothesis will be tested by determining if immunogenicity after TIV or TIV/CLDC vaccination of these elderly subjects can be predicted by the capacity of their monocytes, B cells, and T cells to signal after engagement of receptors for cytokines, e.g., IL-21, innate immune ligands, e.g., CpG DNA, and antigen, i.e., the B-cell and T-cell receptors, as assessed by an improved phospho-flow cytometric assay. In parallel, the frequency of the major subsets of monocytes, B cells, and CD4 and CD8 T- cells will be determined by polychromatic flow cytometry, which will be important in the interpretation of the phospho-flow results. The leukocyte subsets and the stimuli that are most robust predictors of TIV or TIV/CLDC immunogenicity will then be evaluated for their basal and stimulus-dependent levels of mRNA and miRNA expression, focusing on changes that are known to or are likely to influence signaling. Together, these studies will define the predictive value and biological importance of the level of leukocyte subset signaling in generating adaptive immune responses to TIV and TIV/CLDC in the elderly. They will also provide new mechanistic insights as to the role that specific mRNAs and miRNAs play in regulating signaling and activation-induced gene expression involved in vaccine immunogenicity. PUBLIC HEALTH RELEVANCE (provided by applicant): A major limitation in using vaccines to prevent infections, such as influenza, in high-risk populations, such as the elderly, is that it is difficult to predict whether an immunization will be effective in sufficiently boosting the immune response to protect that individual from the infectious disease. This research will identify tests of the function of white blood cells of the immune system that can be used to predict whether vaccination of an elderly individual for influenza will be likely to sufficiently stimulate the immune system to provide that individual with protection from infection. These tests will help use current vaccines more effectively, and will also help in the development of a new, more potent vaccine for influenza that uses an adjuvant, a substance added to vaccines to boost the immune response.